阅读说明：本技术 检测rs9273471位点多态性的试剂及其应用 (Reagent for detecting rs9273471 site polymorphism and application thereof ) 是由 周智广 谢志国 黄干 李霞 罗说明 林健 肖扬 夏影 于 2020-06-17 设计创作，主要内容包括：本发明属于分子生物学和医学领域,涉及一种检测rs9273471位点多态性的试剂及其应用。通过该位点基因型的检测结果来预测成人隐匿性自身免疫糖尿病(LADA)患病风险或者预测区分LADA和2型糖尿病(T2DM)。本发明还公开了相应的检测试剂盒,该试剂盒含有扩增rs9273471位点的引物以及检测该位点基因型的引物。利用本发明检测rs9273471位点的基因型,方法简单易行,快速高效,成本低廉,为LADA的诊断和治疗提供了一个简捷的新途径。(The invention belongs to the fields of molecular biology and medicine, and relates to a reagent for detecting rs9273471 site polymorphism and application thereof. The detection result of the locus genotype is used for predicting the risk of Latent Autoimmune Diabetes (LADA) of adults or predicting and distinguishing LADA from type 2 diabetes (T2 DM). The invention also discloses a corresponding detection kit, which contains a primer for amplifying the rs9273471 locus and a primer for detecting the genotype of the locus. The method for detecting the genotype of the rs9273471 locus is simple, easy, rapid, efficient and low in cost, and provides a simple new way for diagnosis and treatment of LADA.)

1. Application of the reagent for detecting rs9273471 locus polymorphism in preparing a preparation for predicting LADA disease risk.

2. The use as claimed in claim 1, wherein the rs9273471 site polymorphism is: AA, AG or GG.

3. The use of claim 1 or 2, wherein when the genotype at the rs9273471 locus of the test subject is AA, the risk of LADA is higher than that when the genotype is GG.

4. Application of a reagent for detecting rs9273471 site polymorphism in preparation of prediction preparations for distinguishing LADA from T2 DM.

5. The use as claimed in claim 4, wherein the rs9273471 site polymorphism is: AA, AG or GG.

6. The use of claim 4 or 5, wherein the probability of the genotype at site rs9273471 suffering from LADA instead of T2DM in the sequence AA > AG > GG genotype in distinguishing LADA from T2DM is AA > AG > GG genotype.

7. A kit for predicting the risk of LADA disease or for predicting the differentiation between LADA and T2DM, characterized by: is a reagent for detecting rs9273471 site polymorphism.

8. The kit of claim 7, wherein:

comprises a primer for specifically amplifying the rs9273471 locus and a sequencing primer.

9. The kit of claim 8, wherein: the primer sequence for specifically amplifying the rs9273471 site is as follows:

F：TGCATCAAGCTGAAGTTCTGTG；R：GGTGGGGATGAAAGGAGATG。

10. the kit of claim 8, wherein:

the sequence of the sequencing primer is as follows: TGGGAATTCTGGGCAGG are provided.

The technical field is as follows:

the invention relates to the fields of molecular biology and medicine, in particular to a detection reagent for a single nucleotide polymorphism locus rs9273471 of a human HLA gene and application thereof in preparing a reagent for assessing LADA-related risks.

Background art:

latent autoimmune diabetes mellitus (LADA) in adults is an autoimmune diabetes that is developed by adults, has autoantibodies associated with diabetes, and does not require insulin treatment for a certain period of time after diagnosis, and is the most common one of adult-type autoimmune diabetes and possibly the most common autoimmune diabetes. In terms of immunity, glutamate decarboxylase autoantibodies are the most common autoantibodies in adult diabetics.

LADA, a specific subtype of type 1 diabetes (T1DM), is also a T cell mediated autoimmune disease associated with HLA class II antigen abnormally expressed on the cell surface of pancreatic islet β, HLA gene, also called human major histocompatibility complex, located in the short arm of the 6 th chromosome pair, with nearly 60 loci on the entire complex, and based on the difference in the characteristics of the encoding molecules, the genes of the entire complex can be classified into three types, i.e., class I, class II, and class III.the class II gene region, which is located near the centromere, is a region with the most complex structure, and is mainly composed of three subregions of DR, DQ, and DP, each subregion has several loci, wherein rs9273471, which is the locus of the DQ subregion on the class II gene, CD4⁺The T cell surface receptor of (2) is combined with a compound formed by HLA class II molecules and antigen peptides, and stimulates helper T cells to secrete cytokines and the like, thereby participating in the pathogenesis process of LADA. Infiltration of islets by T cells and antigen presenting cells is a lengthy process, resulting in the onset of LADA that can occur months to years after an immune response occurs.

Diabetes is a multifactorial disease caused by a complex interaction between environmental and genetic risk factors. Genetic determinants of type 1 diabetes (T1DM) and type 2 diabetes (T2DM) have been extensively studied, while latent autoimmune diabetes in adults (LADA) has been rarely studied. In order to finally realize the treatment of LADA, the research on susceptible genes of LADA and the development of methods, kits and related treatment medicines for detecting the susceptible genes of LADA are urgently needed in the field. Furthermore, LADA patients are often misdiagnosed with type 2 diabetes due to similar clinical symptoms. In order to distinguish LADA from other types of diabetes, more precise treatment of LADA patients, LADA detection and treatment requires specialized strategies.

The invention content is as follows:

the invention mainly aims to provide application of a reagent for detecting rs9273471 site polymorphism in preparation of a preparation for predicting LADA disease risk. The polymorphic locus is found to be related to the LADA disease risk for the first time, the genotype of the polymorphic locus is used for judging the LADA disease risk, and the polymorphic locus has high prediction accuracy and high reliability.

Further, the rs9273471 site polymorphism is as follows: AA, AG or GG.

The invention discovers that the number of normal people in people carrying rs9273471GG genotype is far higher than that of patients with LADA (mammary gland activator), and the risk of suffering LADA of GG genotype patients is low, and the other two genotypes AA and AG take GG genotype as reference.

The invention uses about 500 cases of LADA cases and normal controls to carry out statistical analysis to find that: when the genotype of the locus rs9273471 is AA, the ratio OR of GG relative to the genotype is 4.14, and the p value is less than 0.05, so that the significant difference exists. The risk of the population carrying the rs9273471AA genotype to suffer from LADA is obviously increased and is 4.14 times of the risk of the population carrying the GG genotype to suffer from LADA; when the rs9273471 locus genotype is AG, the ratio OR of GG relative to the genotype is 1.31, but the p value is more than 0.05, and the difference is not significant, so that when the rs9273471 locus genotype is AG, the risk of LADA is not enough to be judged.

Further, the present invention concludes that: when the genotype of the locus rs9273471 of the detection object is AA, the LADA risk is higher than that of GG.

Further, the reagent for detecting rs9273471 site polymorphism can predict the following situations:

(1) for predicting the risk of LADA in the general population not presenting with clinical symptoms of diabetes.

(2) For predicting the risk of LADA in a person who presents clinical symptoms of diabetes but has not yet been diagnosed with diabetes.

(3) For predicting the risk of LADA in patients with confirmed, but not yet confirmed, diabetes.

The second purpose of the invention is to provide an application of a reagent for detecting rs9273471 site polymorphism in preparing a prediction preparation for distinguishing LADA and T2 DM. The polymorphic locus is found for the first time and can be used for predicting and distinguishing LADA and T2DM, the genotype of the polymorphic locus is used for judging the probability of suffering from LADA but not T2DM, the prediction accuracy is high, and the reliability is high.

Further, the rs9273471 site polymorphism is as follows: AA, AG or GG.

Because the number of T2DM patients is far higher than that of LADA patients in the population carrying the rs9273471GG genotype, the probability that the GG genotype is suffered from LADA instead of T2DM is low, and the GG genotype is taken as a reference for the other two genotypes AA and AG.

The invention discovers by carrying out statistical analysis on about 500 cases of each of LADA (mammary epithelial amyloid) cases and T2DM cases:

when the rs9273471 locus genotype is AA, the ratio OR of GG relative to the genotype is 3.45,

when the rs9273471 locus genotype is AG, the ratio OR of GG relative to the genotype is 1.44.

That is, the probability of the people carrying rs9273471AA genotype to suffer from LADA instead of T2DM is obviously increased, and the probability of suffering from LADA instead of T2DM is 3.45 times of GG genotype; the probability of LADA rather than T2DM in people carrying the rs9273471AG genotype is 1.44 times higher than in people with the GG genotype.

Furthermore, when the LADA and the T2DM are distinguished, the probability that the rs9273471 locus genotype suffers from the LADA instead of the T2DM is AA & gtAG & gtGG genotypes in sequence, and the p values are all smaller than 0.05, so that the significant difference is achieved.

The reagent for detecting the rs9273471 site polymorphism can predict the following situations:

(1) for predicting the probability of LADA instead of T2DM in a person who presents clinical symptoms of diabetes but has not yet been diagnosed with diabetes.

(2) For predicting the probability that a patient diagnosed with diabetes, but not specifically who diabetes, will have LADA instead of T2 DM.

(3) For predicting the probability of a patient diagnosed with diabetes, but not yet diagnosed with LADA or T2DM, to have LADA instead of T2 DM.

In the case of the (3) cases, patients with LADA are often misdiagnosed as type 2 diabetes due to similar clinical symptoms, and therefore, the differentiation in this application is medically significant.

It is a third object of the invention to provide a kit for predicting the risk of LADA disease or for distinguishing LADA from T2 DM. The kit is designed based on the newly found SNP sites for predicting the LADA disease risk or distinguishing LADA and T2DM, and has high prediction accuracy and high reliability.

The kit is a reagent for detecting rs9273471 site polymorphism.

Further, the kit comprises:

comprises a primer for specifically amplifying the rs9273471 locus and a sequencing primer.

Further, the sequence of the sequencing primer is preferably: TGGGAATTCTGGGCAGG, see SEQ ID NO. 1; however, the kit of the present invention is not limited to this sequencing sequence.

Further, it is preferable that the amplification primer sequence is: f: TGCATCAAGCTGAAGTTCTGTG, see SEQ ID NO. 2; r: GGTGGGGATGAAAGGAGATG, see SEQ ID NO. 3; however, the kit of the present invention is not limited to the amplification primer sequence.

The LADA susceptibility gene detected by the invention is the genotype of the locus rs9273471 of the DQ subregion of the HLA class II gene. The nucleotide sequence of rs9273471 can be found at website: http:// genome. ucsc. edu/.

The matched detection method comprises the following steps:

(a) extracting genomic DNA in a sample;

(b) PCR amplification is carried out to obtain a product containing rs9273471 site;

(c) sequencing the product and analyzing the genotype of the site.

The detection object of the invention is Asian race, especially Chinese.

The techniques of extracting genomic DNA, amplifying, sequencing and the like involved in the above methods can all adopt conventional operation methods in the field.

Through years of research, the invention proves that the single nucleotide polymorphism of the rs9273471 locus of the DQ subregion of the HLA class II gene is related to the morbidity risk of the LADA for the first time. An alteration in the rs9273471 genotype will lead to an increased risk of development of LADA, wherein the results of association studies show a significant difference in the distribution of rs9273471(GG → AA) in case and control groups (P < 0.05); and the site is used for judging the standard genotype, so that the prediction accuracy is higher. In addition, the invention also discovers that because of the similarity of clinical symptoms, the LADA patients are often misdiagnosed as type 2 diabetes, the polymorphic site is found for the first time and can be used for predicting and distinguishing LADA and T2DM, and the standard of the site for judgment is also genotype, so that the prediction accuracy is high, and the reliability is high; therefore, the LADA can be simply and efficiently distinguished from the type 2 diabetes, and the LADA can be used for more accurately treating patients with the LADA, and has great significance.

The invention can be used for early diagnosis of individual LADA susceptibility, comprising the steps of: (1) extracting the genomic DNA of the sample. (2) And amplifying the genomic DNA of the sample by PCR by using the specific amplification primer of rs9273471 to obtain an amplification product. (3) Designing a sequencing primer, and sequencing the rs9273471 locus of the amplification product. Detecting the genotype of the individual HLA gene rs9273471 locus, wherein the individual rs9273471 has AA genotype, and the susceptibility of LADA is obviously higher than that of the common population; therefore, whether the individual has higher incidence risk of the LADA than the common population or not can be judged, and in addition, the method can be used for predicting and distinguishing two types of diabetes of the LADA and the T2 DM. The invention provides a more simple and convenient method for diagnosing and treating LADA.

The invention also develops a corresponding detection kit based on the discovery, and the kit comprises a specific primer for amplifying the site and a sequencing primer. The method for detecting the genotype of the rs9273471 locus is simple, easy, rapid, efficient and low in cost, and provides a simple and new way for diagnosis and treatment of LADA.

Description of the drawings:

FIG. 1 shows the results of the genomic DNA concentration and quality measurements of the samples of example 1.

FIG. 2 shows that the genomic DNAs of the samples of example 1 were all longer than 10kb by agarose gel electrophoresis.

FIG. 3 shows the result of site amplification at rs9273471 in a part of samples of example 2.

FIG. 4 is a screenshot of the result of the rs9273471 site amplification sequencing in example 2; exemplary graphs of three genotypes; the upper panel is rs9273471AA genotype; middle panel is rs9273471AG genotype; the lower panel shows rs9273471GG genotype.

FIG. 5 is a screenshot of the result of the rs9273471 site amplification sequencing in example 3; the upper panel is rs9273471AA genotype; middle panel is rs9273471AG genotype; the lower panel shows rs9273471GG genotype.

The specific implementation mode is as follows:

the invention is further illustrated below with reference to specific embodiments. These embodiments are merely illustrative of the present invention and do not limit the scope of the present invention. The experimental procedures, for which no specific conditions are indicated, are according to the usual conditions or according to the conditions recommended by the manufacturer.